title: A role for zinc in regulating hypoxia-induced contractile events in pulmonary endothelium.
abstract: We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31-8220 and a dominant-negative construct of PKC-ε attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.
authors.affiliation: Department of Cell Biology, The University of Pittsburgh, Pennsylvania 15219, USA.
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Bernal
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Bauer
authors.forename: Rong
authors.initials: R
authors.lastname: Cao
authors.forename: Salony
authors.initials: S
authors.lastname: Maniar
authors.forename: Mackenzie
authors.initials: M
authors.lastname: Mosher
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Qiming Jane
authors.initials: QJ
authors.lastname: Wang
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Glorioso
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Pitt
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.forename: Claudette M
authors.initials: CM
authors.lastname: St Croix
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D000860:Hypoxia; D009119:Muscle Contraction / Q000187:drug effects*; D009124:Muscle Proteins / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D012756:Sheep; D015398:Signal Transduction; D022502:Stress Fibers; D015032:Zinc / Q000494:pharmacology*
chemical_list: D000199:Actins; D009124:Muscle Proteins; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; D015032:Zinc